Chlamydia species are the etiological agent for a diverse spectrum of human diseases that include ocular, pulmonary, lymphatic, cardiac and/or genital manifestations. Chlamydia is obligate intracellular pathogens that undergo a complex and unique developmental life cycle that is intrinsically linked to chlamydial pathogenesis. Numerous genes have been identified that are expressed primarily at specific stages of the developmental cycle. A global analysis of chlamydial gene expression during the developmental cycle demonstrates that 22% of the genes are temporally regulated and many of these gene products play key role(s) in the chlamydial developmental cycle and are likely virulence determinants. In prokaryotes, the primary stage of gene regulation is governed by transcriptional initiation. Sigma (G) factors are essential for the initiation of transcription and coordinate the expression of subsets of genes based on the recognition of specific promoter regions. Chlamydia encode three sigma factors, Sigma 68, the primary Sigma factor and two alternative Sigma factors, Sigma 28 and Sigma 54. While data support that Sigma 66 is responsible for the transcription of most constitutively-expressed genes, the role of Sigma 28 and Sigma 54 are currently unknown. This proposal is designed to analyze the hypothesis that Sigma 28 and Sigma 54 direct transcription of developmental stage-specific genes and virulence determinants. These stage-specific genes include but are not limited to; type III secretion systems and effector molecules, transcription factors and DNA compaction proteins. Very little is known about the cis- and trans-acting elements involved in chlamydial gene regulation and data generated during this study will provide a molecular basis for the specific mechanisms of gene regulation in Chlamydia.